Influenza vaccine compositions

ABSTRACT

PCT No. PCT/GB96/02680 Sec. 371 Date Apr. 29, 1998 Sec. 102(e) Date Apr. 29, 1998 PCT Filed Nov. 1, 1996 PCT Pub. No. WO97/16208 PCT Pub. Date May 9, 1997The invention provides a vaccine composition in the form of a kit, comprising a first container containing an antigenic preparation comprising influenza antigen or antigens; and a second container containing an effective adjuvant amount of a chitosan which is a deacetylated chitin which is at least 80% deacetylated. The antigenic preparation in the first container preferably comprises haemagglutinin and neuraminidase influenza antigens.

The invention relates to a vaccine composition in the form of a kitcomprising a first container containing an antigenic preparationcomprising influenza virus antigens and a second container containing amucosal adjuvant. The invention also relates to a method of immunising apatient against influenza by administering the said composition to thepatient, and a method of enhancing the immunogenicity of an influenzaviral antigen, particularly when administered intranasally, byco-administering therewith the said adjuvant.

Current influenza vaccines consist of either inactivated whole virus,disrupted virus (split vaccines) or purified preparations of themembrane glycoproteins haemagglutinin (HA) and neuraminidase (NA)sub-unit vaccines. Haemagglutinin and neuraminidase are the antigens towhich protective antibody responses are directed, haemagglutinin beingthe major protective antigen. Estimates of the efficacy of theseparenterally administered vaccines vary greatly. Such vaccines arebelieved to act primarily by eliciting circulating anti-haemagglutininlgG antibodies that transudate into the lower respiratory tract.

M. L. Clements et al, J. Clinical Microbiology 24, 157-160, 1986, havepreviously reported that both secretory IgA and serum lgG participate inimmunity to influenza virus. Moreover, in mice, a number of publishedstudies have demonstrated the importance of respiratory IgA toprotection against influenza infection. It has also been found that anadvantage of stimulating a local IgA response to influenza is that it isoften of a broader specificity than the serum response and thus canprovide cross-protection against viruses possessing haemagglutininmolecules different from those present in the vaccine. Accordingly,influenza vaccines that elicit both local secretory and serumanti-haemagglutinin responses should provide superior immunity tocurrent vaccines. However, parenteral vaccination (intramuscular,sub-cutaneous etc) is not effective at eliciting local antibodyproduction, if there has been no previous mucosal exposure (e.g.infection). In order to stimulate the mucosal immune system, the vaccinemust be applied topically to a mucosal surface.

Mucosal administration of influenza vaccine would have a number ofadvantages over traditional parenteral immunisation regimes. Paramountamongst these are more effective stimulation of the local mucosal immunesystem of the respiratory tract and the likelihood that vaccine uptakerates would be increased because the fear and discomfort associated withinjections would be avoided. Accordingly, a number of attempts have beenmade to develop mucosal influenza vaccines. A drawback however is thatinactivated vaccines are often poorly immunogenic when given mucosally.For example, Kuno-sakai et al (vaccine 12:1303-1310, (1994) have shownthat administration of inactivated vaccine to humans gave strong mucosaland serum antibody responses and was effective in preventing infectionby live vaccine virus. However, in order to achieve such results,Kuno-sakai et al administered three times the commercially availabledose, an approach which is not considered to be commercially viable. Inorder to overcome this problem, different approaches to improving theimmunogenicity of flu vaccines given orally or intranasally haveincluded the use of the B sub-unit of cholera toxin (CTB) as an adjuvant(see S. Tamura et al, vaccine, 6, 409, (1988), encapsulation of thevaccine in a variety of microspheres (see Z. Moldoveanu et al,J.lnf.Dis. 167, 85-90 (1993), and the use of live attenuated strains(see H. F. Maassab et al, vaccines, Plotkin S. A and Mortimer F. A Jr(eds) W. B. Saunders Philadelphia p435 (1993). To date however, nopractical means of enhancing the immunogenicity of mucosallyadministered flu vaccines has been developed.

It has now been found by the Applicants that by administering influenzaantigens such as the haemagglutinin and neuraminidase antigens incombination with a particular chitosan derivative, it is possible toachieve good IgG and good IgA responses. More particularly , the presentApplicants have now found that if a chitosan is co-administeredintranasally with influenza antigens such as the neuraminidase andhaemagglutinin antigens, or if a host is predosed with a chitosan priorto administration of influenza antigens, good systemic and local immuneresponses are produced.

Chitosans are derivatives of chitin or poly-N-acetyl-D-glucosamine inwhich the greater proportion of the N-acetyl groups have been removedthrough hydrolysis.

Chitosans have previously been used in pharmaceutical formulations andare disclosed in EP-A-0460020 as mucosal absorption enhancers, However,EP-A-0460020 does not disclose or suggest that the chitosan couldprovide an adjuvant effect when administered in a vaccine composition.

Accordingly, in a first aspect, the invention provides a vaccinecomposition in the form of a kit comprising a first container containingan antigenic preparation comprising influenza antigen or antigens; and asecond container containing an effective adjuvant amount of a chitosan.

The influenza antigens can be, for example, inactivated whole influenzavirus, or purified or partially purified antigens. In an alternativeembodiment, the influenza antigens can be present in a carrier molecule,for example as part of a fusion protein.

The antigenic preparation preferably contains haemagglutinin andneuraminidase influenza virus antigens. More particularly, the antigenicpreparation preferably is inactivated whole influenza virus (WIV) or acomposition containing purified haemagglutinin and neuraminidaseinfluenza virus antigens.

Where a purified haemagglutinin and meuraminidase composition isemployed, it is preferred that the purified haemagglutinin andneuraminidase antigens are present in the form of rosettes. The rosettespreferably are particles with a radius in the range 10 to 25 nanometres.It is preferred that the rosettes are substantially free of lipid and,moreover, it is preferred that the purified haemagglutinin andneuraminidase antigen preparation as a whole is substantially free oflipids.

An example of a haemagglutinin/neuraminidase preparation suitable foruse in the compositions of the present invention is the "Fluvirin"product manufactured and sold by Evans Medical Limited of Speke,Merseyside, United Kingdom, and see also S. Renfrey and A. Watts,Vaccine, 1994, Volume 12, Number 8, pp 747-752.

Whole influenza virus vaccines can be prepared from killed influenzavirus strains in known fashion. As will be appreciated, such inactivatedwhole virus preparations are a precursor to the purified preparationsdescribed above.

The vaccine compositions of the invention are preferably adapted fordelivery to a mucosal surface, and most preferably are adapted for oralor intranasal delivery.

Preferably the chitosan used in the compositions of the invention iswater-soluble.

The chitosan may advantageously be a deacetylated chitin which is atleast 75% and preferably is at least 80% deacetylated. More preferablythe chitosan is at least 85% de-acetylated, and particularly preferablyis 88% to 90% de-acetylated.

A particular de-acetylated chitosan is the "Sea Cure G210" chitosan(Poly D-glucosamine hydroglutamate) available from Pronova Biopolymer ofDrammen, Norway. This chitosan which is in the form of its glutamatederivative, has >80% deacetylation.

In a further aspect, the invention provides a method of immunising ahost against infection with influenza, which method comprisesadministering to the host:

(i) from a first container an antigenic preparation comprising aninfluenza virus antigen or antigens as hereinbefore defined and

(ii) from a second container an effective adjuvant amount of a chitosanas hereinbefore defined.

The contents of the first and second containers can be administered atsubstantially the same time, ie simultaneously or within a few minutesof each other, or they may be administered at spaced intervals. Forexample, the host may be pre-dosed with the chitosan and the antigenicpreparation administered subsequently.

The first and second containers can be entirely separate or can beconstituted by separate chambers of the same applicator device. Wherethe containers are separate, they could be provided in the form of a kitcomprising first and second aerosol dispensers, or first and secondsyringes, by way of example. Where the first and second containers formpart of the same applicator, they could for example, be defined by twobarrels of a multibarrel syringe. Such applicators containing aninfluenza antigenic preparation in one chamber and a chitosancomposition in another chamber form a further aspect of the invention.

In a further aspect, the invention provides a method of inducing aprotective mucosal immune response and a systemic immune response byadministering to a patient

(i) from a first container an antigenic preparation comprising aninfluenza virus antigen or antigens as hereinbefore defined and

(ii) from a second container an effective adjuvant amount of a chitosanas hereinbefore defined.

In a still further aspect, the invention provides a method of inducing aprotective IgA mucosal immune response and an IgG systemic immuneresponse by administering to a patient

(i) from a first container an antigenic preparation comprising aninfluenza virus antigen or antigens, and

(ii) from a second container an effective adjuvant amount of a chitosan,

wherein the antigenic preparation and chitosan are as hereinbeforedefined.

In another further aspect, the invention provides a method of enhancingthe immune response of influenza virus antigens by co-administeringtherewith from a separate container a chitosan as hereinbefore defined.

The compositions of the invention can be presented for administration bya number of routes but preferably they are presented for administrationmucosally, and in particular intranasally. Intranasal compositions canbe formulated for example as liquids or dry powders, for administrationas aerosols or drops.

Compositions for administration as nasal drops may contain one or moreexcipients of the type usually included in such compositions, forexample preservatives, viscosity adjusting agents, tonicity adjustingagents, buffering agents and the like.

The present invention also contemplates the provision of means fordispensing intranasal formulations of influenza virus antigens andchitosan. A dispensing device may, for example, take the form of anaerosol delivery system, and may be arranged to dispense only a singledose, or a multiplicity of doses. Thus the first and second containersmay be adapted for use in an aerosol dispensing device.

The antigenic preparation will be administered to the patient in anamount effective to stimulate a protective immune response in thepatient. For example, the antigenic preparation may be administered tohumans in one or more doses, each dose containing 1-250 microgrammes andmore preferably 1-50 microgrammes of protein prepared from each virusstrain. For example, where haemagglutinin and neuraminidase preparationsare prepared from three virus strains, e.g. 2×Influenza A and1×Influenza B, a total dose of viral protein administered could be inthe range 3-150 microgrammes.

The invention will now be illustrated, but not limited, by reference tothe drawings and the following examples.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the serum IgG anti-haemagglutinin response in miceimmunised with inactivated whole influenza A/Texas (WIV). Each barrepresents the log geometric mean titre of five mice. The error barsrepresent 1 standard error of the geometric mean.

FIG. 2 illustrates the nasal IgA anti-influenza A/Texas/WIV response inmice immunised with WIV. As with FIG. 1, each bar represents thegeometric mean titre of four mice, and the error bars represent 1standard error of the geometric mean.

FIGS. 3a and 3b illustrate the determination of nasal and pulmonary WIVspecific antibody secreting cells (ASC) in mice immunised with WIV,using ELISPOT.

EXAMPLE 1 PREPARATION OF CHITOSAN FORMULATION

A solution of 1 % chitosan glutamate, a medium viscosity de-acetylatedchitin having greater than 80% deacetylation, was prepared by dissolvingthe chitosan glutamate in 0.5% W/V sodium chloride by stirring at roomtemperature for 48 to 60 hours. The pH of the resulting solution wasincreased to 5.8-6.0 using 0.1M Na₂ HPO₄. The grade of chitosanglutamate used was a medium viscosity grade sold under the trade name"Sea Cure+G210" (Product Code 27150210), by Pronova Biopolymer ofDrammen, Norway.

EXAMPLE 2 PREPARATION OF INFLUENZA ANTIGENIC FORMULATIONS

Purified Surface Antigen (PSA)

2A. Influenza purified surface antigen (PSA) containing InfluenzaA/Texas protein, available from Evans Medical Limited, Speke,Merseyside, United Kingdom, was made up in phosphate buffered saline(PBS) to give a protein concentration of approximately 1 mg/ml. The PSAconsists almost entirely of the spike protein haemagglutinin (HA),although it does contain some neuraminidase.

WHOLE INFLUENZA VACCINE (WIV)

2B. Whole influenza vaccine (WIV) of the A/Texas strain was made up to1mg/mi protein concentration in PBS.

EXAMPLE 3

Preparation of a Chitosan/Influenza Admixture

A 1:1 mixture of the chitosan glutamate solution and the PSA or WIVsolution was prepared to give an intranasal vaccine compositioncontaining 0.5% chitosan glutamate and 0.5 mg/ml influenza antigen.

Control solutions containing the same concentrations of WIV or PSA butnot chitosan glutamate, were also prepared.

EXAMPLE 4

Mice Immunisation Studies

The compositions prepared as described in the Examples above wereadministered to groups of five adult (6-8 weeks) female BALB/c mice asfollows:

    ______________________________________                                                                 Dose/mice                                                                      Group Formulation (total protein)                   ______________________________________                                        A         WIV + chitosan 10 μg                                                (separate administration)                                                    B WIV + chitosan 10 μg                                                      (admixture)                                                                  C PSA + chitosan 10 μg                                                      (separate administration)                                                    D PSA + chitosan 10 μg                                                      (admixture)                                                                  E WIV (A/Texas) 10 μg                                                      F PSA (A/Texas) 10 μg                                                    ______________________________________                                    

Immunisations were given intranasally in 20 μl volumes (10 μl pernostril) to the mice in groups B, D, E, F. The group B and D micereceived the preparations described in Example 3 above, whilst the groupE and F control mice received the formulations of Examples 2B and 2Arespectively and no chitosan. The mice in groups A and C first received5 μl per nostril of the chitosan composition of Example 1 and then,following a recovery time of 4 minutes, were given 5 μl per nostril ofthe influenza formulations of Examples 2B and 2A respectively. Theimmunisation and sampling regime used is shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Immunisation and sampling regime                                                  Immunisation                                                                             Day          Sample                                                                              Day                                         ______________________________________                                        1           0           1       21                                              2 28 2 42                                                                     3 56 3 74                                                                   ______________________________________                                    

At sampling points 1 and 2, all mice from each group were sample bled.Sampling at sample point 3 was performed by cardiac puncture, followingwhich the heads of the mice were removed and their nasal passageslavaged with 1 ml PBS+1 % bovine serum albumin.

Antibody and ELISPOT Assays

The immune responses stimulated by the immunisation regimes describedabove were analysed in conventional fashion using the ELISPOT and ELISAtechniques. Thus, an Enzyme Linked Immunosorbant Assay (ELISA) was usedto determine the serum IgG anti-A/Texas response, and the results areshown in FIG. 1 and Table 2 below. The ELISA technique was also used todetermine the nasal IgA anti-A/Texas response and the results of thisassay are shown in FIG. 2 and Table 3 below. In all assays wholeinfluenza vaccine (WIV) was used as antigen. ELISA endpoint titres werecalculated as sample dilutions yielding a result of 0.3 OD units for IgGand 0.1 OD units for IgA assay after background control (without sample)subtraction.

RESULTS

                                      TABLE 2                                     __________________________________________________________________________    Serum anti-WIV serum response (Table 2 and FIG. 1)                                       Post-Dose 1   Post-Dose 2  Post-Dose 3                             Group      Seroconversion*                                                                        GMT.sup.+                                                                          Seroconversion                                                                        GMT  Seroconversion                                                                        GMT                             __________________________________________________________________________    A.                                                                              WIV + chitosan                                                                         5/5      3005 5/5     29708                                                                              5/5     44338                              (separate)                                                                   B. WIV + chitosan 5/5 2038 5/5 41426 5/5 63157                                 (admixture)                                                                  C. PSA + chitosan 4/5  271 5/5  9314 5/5 30599                                 (separate)                                                                   D. PSA + chitosan 5/5 1017 5/5 69331 5/5 144224                                (admixture)                                                                  E. WIV (A/Texas) 5/5  499 5/5  3800 5/5  6044                                 F. PSA (A/Texas) 0/5  49 2/5   89 4/4  428                                  __________________________________________________________________________     *No. positive/No. tested                                                      .sup.+ Geometric Mean Titre                                              

Nasal IgA anti-WIV response

TABLE 3

Nasal IgA anti-A/Texas response EU/μg total IgA (Individualresponses--geometric mean+sem)

    ______________________________________                                                     Individual IgA Geometric Mean                                      Group Responses (EU/μg) (EU/μg)                                       ______________________________________                                        A.  WIV + chitosan                                                                             342 351 405 240 246                                                                          310 ± 65                                      (separate)                                                                   B. WIV + chitosan 263 100 328 447 250 249 ± 126                             (admixture)                                                                  C. PSA + chitosan 74 265 142 14 100 83 ± 52                                 (separate)                                                                   D. PSA + chitosan 283 199 289 193 490 274 ± 92                              (admixture)                                                                  E. WIV (A/Texas) 52 89 156 67 254 104 ± 61                                 F. PSA (A/Texas) 7 9 5 4 6 ± 2                                           ______________________________________                                    

Antibody Secreting Cells (ASC) Specific for WIV in the Nasal andPulmonary Mucosa

Lymphocytes were isolated from the nasal mucosa and lung parenchyma ofgroups of five mice at the third sampling point. Lymphocytes fromindividual mice were pooled and assayed for cells secreting IgA, IgG andIgM anti-flu antibodies using ELISPOT. The results are shown in FIGS. 3aand 3b. In FIGS. 3a and 3b, the antibody secreting cells detected by theELISPOT assay are expressed as the number of positive cells/10⁷ totalcells (lymphocytes). FIG. 3a illustrates the effect of chitosan on thenasal and pulmonary immunogenicity of the PSA preparation of Example 2Awhereas FIG. 3b illustrates the effect of chitosan on the nasal andpulmonary immunogenicity of the WIV preparation of Example 2B. Theresults shown in FIGS. 3a and 3b illustrate that chitosan enhances theIgA ASC response to both PSA and WIV, the greater enhancement beingobserved with PSA. The strongest ASC responses were generated in thenasal mucosa with IgA ASC being dominant. Pulmonary responses werepredominantly IgG secreting cells.

In general, greater ASC responses were induced by both PSA and WIV whenthey were formulated with chitosan, than when the chitosan wasadministered first. In the case of the IgA ASC response, the formulationcontaining both PSA and chitosan produced a greater response than wasobtained when chitosan was administered first. However, there was nosignificant difference between the IgA ASC responses induced by theformulation containing both WIV and chitosan, and WIV preceded by thechitosan.

The results set out above illustrate that good immune responses can beobtained by administering influenza antigens and chitosans separatelyvia the mucosal route. This offers an advantage in that it enables thepotential problems associated with administration by injection to beavoided.

The aforementioned examples are merely exemplary of the presentinvention and are not intended in any way to limit the scope of theinvention which is defined solely by the claims appended hereto.

What is claimed is:
 1. A kit for providing an influenza vaccine, saidkit comprising:a first container containing an antigenic preparationcomprising influenza antigen or antigens; and a second containercontaining an effective adjuvant amount of a chitosan, wherein thechitosan is a deacetylated chitin which is at least 80% deacetylated. 2.A kit according to claim 1 wherein the antigenic preparation in thefirst container comprises haemagglutinin and neuraminidase influenzaantigens.
 3. A kit according to claim 2 wherein the antigenicpreparation in the first container is a whole influenza viruspreparation.
 4. A kit according to claim 2 wherein the antigenicpreparation in the first container comprises purified haemagglutinin andneuraminidase influenza antigens.
 5. A kit according to claim 4 whereinthe purified haemagglutinin and neuraminidase influenza antigens arepresent in the form of rosettes having a radius in the range 10 to 25nanometers.
 6. A kit according to claim 1 wherein the antigenicpreparation and the chitosan are adapted for mucosal administration. 7.A kit according to claim 6 wherein the antigenic preparation and thechitosan are adapted for intranasal administration.
 8. A kit accordingto claim 6 wherein the antigenic preparation and the chitosan areadapted for oral administration.
 9. A kit according to claim 1 whereinthe chitosan is at least 85% deacetylated.
 10. A kit according to claim9 wherein the chitosan is 88% to 90% deacetylated.
 11. A kit accordingto claim 1 wherein the chitosan is water-soluble.
 12. A kit according toclaim 1 wherein the chitosan is present in the second container in asolution which has a pH in the range 5.5 to 6.5.
 13. A kit according toclaim 12 wherein the pH of the chitosan solution is approximately 5.8-6.14. A kit according to claim 1 wherein the first and second containersare adapted to cooperate with a dispenser for administering the contentsthereof intranasally.
 15. A kit according to claim 1 wherein the firstand second containers are constituted by separate chambers in the samedispensing device.
 16. A kit according to claim 15 wherein the first andsecond containers are constituted by two barrels of a multi-barrelsyringe.
 17. A pharmaceutical product for providing an intranasalinfluenza vaccine, said product comprising:a dispensing device adaptedto deliver an intranasal pharmaceutical dosage; and a kit for providingan intranasal influenza vaccine, said kit comprising: a first containercontaining an antigenic preparation comprising influenza antigen orantigens; and a second container containing an effective adjuvant amountof a chitosan, wherein the chitosan is a deacetylated chitin which is atleast 80% deacetylated, wherein said antigenic preparation and saidchitosan are adapted for intranasal administration.
 18. A pharmaceuticalproduct according to claim 17 wherein the dispensing device is anaerosol delivery system.
 19. A method of immunising a host againstinfection with influenza, which method comprises separatelyadministering to the host:(i) from a first container an antigenicpreparation comprising an influenza virus antigen or antigens, and (ii)from a second container an effective adjuvant amount of a chitosanwherein the chitosan is a deacetylated chitin which is at least 80%deacetylated.
 20. A method of enhancing a protective IgA mucosal immuneresponse and an IgG systemic immune response by administering to apatient(i) from a first container an antigenic preparation comprising aninfluenza virus antigen or antigens, and (ii) from a second container aneffective adjuvant amount of a chitosan, wherein the chitosan is adeacetylated chitin which is at least 80% deacetylated.
 21. A method ofenhancing the immune response of influenza virus antigens, whenadministered to a patient intranasally, by co-administering therewithfrom a separate container a chitosan, wherein the chitosan is adeacetylated chitin which is at least 80% deacetylated.